Inflammatory Cytokine-induced Expression of Vasohibin-1 by Rheumatoid Synovial Fibroblasts

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1 Inflammatory Cytokine-induced Expression of Vasohibin- by Rheumatoid Synovial Fibroblasts a b c a a a a d e a* a a b c d e

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5 December 9 Vasohibin- and Rheumatoid Arthritis 5 A C Vasohibin- score Vasohibin- staining score B 6 Inflammation score 8 Fig. A, Comparison of the histologic features of synovial samples derived from patients with rheumatoid arthritis () and osteoarthritis (). Immunohistochemical analysis was performed to detect vasohibin- in and synovial samples. In samples, immunoreactivity for vasohibin- was observed in a few endothelial cells (arrows). In samples, immunoreactivity for vasohibin- was observed in synovial lining cells (arrowheads), endothelial cells (arrows), synovial fibroblasts, and infiltrating inflammatory cells. In samples, the inflammation score was (synovial lining cell hyperplasia, cellular infiltration, fibrosis of the lining cell layer ), the vasohibin- score, and the VEGF score. In the samples, the inflammation score was (synovial lining cell hyperplasia, cellular infiltration, fibrosis of the lining cell layer ), the vasohibin- score, and the VEGF score ; B, The vasohibin- score of synovial tissues. The vasohibin- scores of and synovial tissues were. ±. and.7 ±.7, respectively (.8), as determined by the method described in the Materials and Methods. The values in each column are the means ± SD; C, Correlation between inflammation scores and vasohibin- scores. The inflammation score was also positively correlated with the vasohibin- score in the synovium (.8,., ), but not in the synovium ( =.8,.9, 9). moxic condition down-regulated vasohibin- expression at 8h (.). Stimulation by the combination of IL-β (ng/ml) and TNF-α (ng/ml) under a normoxic condition also down-regulated vasohibin- expression at 8h (.) (Fig. 5A). Under a hypoxic condition, TNF-α (ng/ml) stimulation did not up-regulate vasohibin- expression. However, IL-β (ng/dl) stimulation under a hypoxic condition up- regulated vasohibin- expression at h (.5), and significantly down-regulated vasohibin- expression at 8h (.). Stimulation by the combination of IL-β (ng/ml) and TNF-α (ng/ml) under a hypoxic condition further enhanced the mrna expression of vasohibin- (Fig. 5B) up to h (.5; Fig. 5B). -

6 Miyake et al. 5 Acta Med. Okayama Vol. 6, No. 6 A B C Vasohibin- score VEGF score VEGF score Fig. A, Immunohistochemistry for VEGF in synovial samples from patients with and. In samples, immunoreactivity for vasohibin- was observed in a few synovial lining cells (arrowheads) or endothelial cells (arrows). In samples, immunoreactivity for VEGF was strongly observed in infiltrating cells, synovial lining cells (arrowheads), synovial fibroblasts, and endothelial cells (arrows); B, The VEGF score of synovial tissues. The VEGF scores of and synovial tissues were.7 ±.5 and. ±.88, respectively, and these values were significantly different (.), as determined by the method described in the Materials and Methods. The values in each column are the means ± SD; C, Correlation between VEGF scores and vasohibin- scores in the synovium ( ). The VEGF score was positively correlated with the vasohibin- score (.76,.5). We next examined the levels of VEGF mrna in SFs by stimulation with cytokines under a normoxic or hypoxic condition. VEGF mrna expression was not up-regulated by TNF-α (ng/ml), but was up-regulated by IL-β (ng/ml) under a normoxic condition. Stimulation of the cells by the combination of IL-β (ng/ml) and TNF-α (ng/ml) under a normoxic condition also enhanced mrna expression of VEGF (Fig. 6A). VEGF expression was markedly up-regulated by TNF-α (ng/ml) or IL-β (ng/ml) under a hypoxic condition. Stimulation of the cells by the combination of IL-β (ng/ ml) and TNF-α (ng/ml) under a hypoxic condition also markedly enhanced the mrna expression of VEGF (Fig. 6B). Discussion Angiogenesis plays a key role in normal vascular development, is a decisive factor in cancer, wound

7 December 9 Vasohibin- and Rheumatoid Arthritis 55 A C Vasohibin- score The number of CD(+) microvessels B The number of CD(+) microvessels 6 Fig. A, Immunohistochemistry for CD. CD-positive endothelial cells were observed in blood vessels (arrows); B, CD( ) microvessel densities (vessel number per field). Microvessel densities were not statistically different between the and synovium (9. ±.9 and.5 ±., respectively;.); C, Correlation between the CD( ) microvessel density (counts per field) and vasohibin- score. The microvessel density was not statistically correlated with the inflammation score in (.5,.69). healing, and inflammation [5], and is regulated by the local balance between angiogenic stimulators and inhibitors. As the pathogenesis of is highly influenced by angiogenesis in the process of forming and maintaining inflammatory synovial tissues, the application of angiogenesis inhibitors for the treatment of has been expected. A number of angiogenesis inhibitors have been investigated and identified, including angiostatin, endostatin, platelet factor- (PF), thrombospondin- (TSP-) [6] and tumstatin [7]. Vasohibin- is a newly identified negative feedback regulator of angiogenesis. In humans, the levels of vasohibin- have been investigated in angiogenesis- associated disorders such as endometrial cancer, or in choroidal neovascular membranes [5, 8], and a potential association with disease activity was demonstrated. The results of the immunohistochemistry in the present study suggested that vasohibin- is expressed in synovial lining cells, endothelial cells, and synovial fibroblasts in synovial tissue. The intensity of the immunoreactivity for vasohibin- was correlated with the inflammation score and VEGF score, and was significantly higher in the synovium than the synovium. In fact, Tamaki. demonstrated an association between vasohibin- expression and inflammation in human breast lesions

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